NATURE BIOTECHNOLOGY

NEWS 27 FEBRUARY 2020.

Charlotte Harrison

Coronavirus puts drug repurposing on the fast track

Existing antivirals and knowledge gained from the SARS and MERS outbreaks gain traction as the fastest route
to fight the current coronavirus epidemic.

China’s biotech companies have been gearing up to repurpose existing drugs, approved in the West for other
viruses, as treatments for the coronavirus outbreak originating in Wuhan.

Last month, Hangzhou-based Ascletis Pharma applied to the Chinese authorities to test two HIV protease
inhibitors (ritonavir and ASC09) in clinical trials to treat COVID-19, the illness caused by the new coronavirus
(Table 1). And Suzhou-based BrightGene Bio-Medical Technology announced in early February that it would
begin to manufacture Gilead Sciences' remdesivir (GS-5734), a broad-spectrum investigational antiviral, as a
treatment for coronavirus infection. Remdesivir, originally developed to treat Ebola virus and then dropped,
will also be tested by Gilead in partnership with Chinese health authorities in randomized, controlled trials.
“The general genomic layout and the general replication kinetics and the biology of the MERS, SARS and [SARS-
CoV-2] viruses are very similar, so testing drugs which target relatively generic parts of these coronaviruses is
a logical step,” says Vincent Munster, chief, Viral Ecology Unit, US National Institute of Health. Testing
therapies approved for other indications also makes senses, as these drugs are already mass produced and
available on a large scale.

From the start of the COVID-19 outbreak, medical practitioners have followed China’s guidelines set up in
January and treated hospitalized patients with α-interferon combined with the repurposed drug Kaletra, an
approved cocktail of the HIV protease inhibitors ritonavir and lopinavir. The World Health Organization has
noted that this combination could provide some clinical benefit. Kaletra, manufactured by AbbVie, is also being
tested in other combinations, with repurposed drugs known to target parts of the replication machinery of
other viruses that are similar to those in SARS-CoV-2 — for instance, with the guanosine analog and RNA
synthesis inhibitor ribavirin, with reverse transcriptase inhibitors (emtricitabine/tenofovir alafenamide
fumarate) or with Moscow-based Pharmstandard’s membrane fusion inhibitor umifenovir. Umifenovir is also
in trials as a single agent.

SARS-CoV-2 is an enveloped, positive-sense, single-stranded RNA β-coronavirus similar to the severe acute
respiratory syndrome (SARS) and Middle East respiratory syndrome (MERS) viruses. Potential antiviral targets
encoded by the viral genome include non-structural proteins (e.g., 3-chymotrypsin-like protease, papain-like
protease, RNA-dependent RNA polymerase and its helicase), structural proteins (e.g., the capsid spike
glycoprotein) and accessory proteins. Kaletra is thought to inhibit the 3-chymotrypsin-like protease of the
SARS and MERS coronaviruses and was associated with improved clinical outcomes in a trial against SARS.
Ascletis also reported that a patient with COVID-19 improved rapidly when given this HIV protease inhibitor
combination.

But Erik De Clercq, of the Rega Institute for Medical Research in Leuven, Belgium, says that in searching for or
designing effective drugs against COVID-19: “We should stay away from antivirals known to be acting at targets
not playing a role in the replication of coronaviruses.” Such drugs include penciclovir, which is targeted at the
herpesvirus DNA polymerase, and lopinavir/ritonavir, which are targeted at the HIV protease. Instead, he
would favor targeting a virus-specific protein such as the RNA-dependent RNA polymerase, noting that
coronaviruses do not contain or use a reverse transcriptase. George Painter, president of the Emory Institute
for Drug Development, Emory University, is also cautious about the HIV protease inhibitor strategy. “It’s
probably a long shot to go for drug repurposing activity against the coronavirus using HIV drugs; these were
protease inhibitors that were designed specifically for HIV,” he says.

Nonetheless, several clinical trials of Kaletra are underway, either alone or with various combinations of
interferons, guanosine-analog RNA synthesis inhibitors, reverse transcriptase inhibitors or influenza drugs,
such as baloxavir marboxil, oseltamivir and umifenovir (Table 1). These trials are anticipated to read out from
the end of May onwards.

Painter is more optimistic about Gilead’s investigational drug remdesivir, a nucleotide analog antiviral, that
blocks the RNA polymerase of the Ebola virus and so prevents replication. The thinking behind repurposing
remdesivir is that its broad antiviral activity may render it effective against SARS-CoV-2. Indeed, remdesivir is
in two clinical trials that began early February, with an estimated completion date of early April. “Remdesivir
has quite high efficacy across all different coronaviruses and therefore it is one of the prime candidates to
start being tested,” says Munster. The World Health Organization’s R&D Blueprint report released at the end
of January considered remdesivir the most promising candidate to treat COVID-19, on the basis of its broad-
spectrum activity, in vitro and in vivo data for coronaviruses and clinical safety from Ebola virus disease trials.
In vitro studies published in January have shown remdesivir to be active against a clinical isolate of SARS-CoV-
2. Experimental data in mice infected with the related MERS virus also showed the drug was better than a
combination of lopinavir/ritonavir and interferon beta in improving lung function. And the first patient in the
United States with confirmed COVID-19 improved after being treated for one day with remdesivir, although
this could not be directly attributed to the drug’s effect. Since then, remdesivir has been shown to reduce the
severity of disease, virus replication and damage to the lungs in a non-human primate model of MERS.

“Broad-spectrum agents are ideally suited for outbreak situations where we don’t entirely know what we are
dealing with in terms of pathogens,” says Bryan Mounce, assistant professor, Department of Microbiology and
Immunology, Loyola University Chicago. “Although we might not understand all the mechanisms underlying
their antiviral activity, it is important that they have as few side effects as possible,” he adds. A second broad-
spectrum antiviral in trials (Table 1) is Tokyo-based Toyama Chemical’s RNA polymerase inhibitor favipiravir,
approved for use against influenza A and B. In an in vitro study, this compound did not show strong activity
against a clinical isolate of SARS-CoV-2.

Another HIV protease inhibitor, Janssen’s Prezcobix (darunavir and the boosting agent cobicistat), is also under
evaluation. At the end of January, Janssen shipped Prezcobix to China for in vitro testing. In a statement to
Nature Biotechnology, the company said, “We are aware of anecdotal reports of darunavir potentially having
antiviral activity against COVID-19. The company has no in vitro or clinical data to support the use of darunavir
as a treatment for COVID-19.” The recipients of the shipments have registered a clinical trial to test Prezcobix
in combinations.

At least ten clinical trials are testing chloroquine, approved as an antimalarial and autoimmune disease drug.
In vitro, the endosomal acidification fusion inhibitor blocked infection of a clinical isolate of SARS-CoV-2.

Most of the drugs in clinical trials (Table 1) inhibit key components of the coronavirus infection lifecycle. These
include viral entry into the host cell (blocked by umifenovir, chloroquine or interferon), viral replication
(blocked by lopinavir/ritonavir, ASC09 or darunavir/cobicistat, which inhibit the 3C-like protease (3CLpro)) and
viral RNA synthesis (inhibited by remdesivir, favipiravir, emtricitabine/tenofovir alafenamide or ribavirin). The
genomic sequence of the SARS-CoV-2 suggests that there is a high level of sequence similarity between the
SARS-CoV-2, SARS and MERS proteins involved in the replication cycle.

Targeting viral cellular entry via the spike glycoprotein, which mediates the virus–cell receptor interaction, is
another option for repurposing. SARS-CoV-2 uses angiotensin-converting enzyme 2 (ACE2) and the cellular
protease transmembrane protease serine 2 (TMPRSS2) to enter target cells. The marketed TMPRSS2 inhibitor
camostat mesylate blocked cellular entry of the SARS-CoV-2 virus, according to an unpublished preprint. And
the Janus-associated kinase (JAK) inhibitor Olumiant (baricitinib), approved for rheumatoid arthritis, was
identified using machine learning algorithms on the basis of its inhibition of ACE2-mediated endocytosis.
Another JAK inhibitor, Jakafi (ruxolitinib), is in trials (combined with mesenchymal stem cell infusion) for
COVID-19.

The release earlier this month of the high-resolution crystal structure of 3CLpro (Protein Data Bank identifier
6LU7) and a Science paper describing the 3.5-Å-resolution structure of the viral spike S protein in pre-fusion
conformation should also expedite drug discovery efforts. Researchers who determined the 3CLpro structure
reportedly used it to screen for repurposed drugs, identifying the protease inhibitors saquinavir, indinavir,
lopinavir and ritonavir, the proteasome inhibitor carfilzomib, two respiratory syncytial virus drugs, a
schizophrenia medication and an immunosuppressant. Also on the basis of the 3CLpro structure, machine
learning algorithms (generative deep learning) generated several novel potential inhibitors. Turning these into
drugs would require a very lengthy drug development process, which would probably be longer than the
current outbreak. One of the authors of the preprint, Alex Zhavoronkov, CEO at Insilico Medicine, Hong Kong,
suggests that finding similarities between these novel structures and known drugs could be an option for
repurposing — and here the speed of machine learning searches could be particularly useful in epidemic
settings.

But even without SARS-CoV-2 structures one can do virtual screening. “For a first approximation, one can work
with the enzyme structures that we have from SARS coronavirus,” says Rolf Hilgenfeld, Institute of
Biochemistry, University of Lübeck, Germany. “Of course, there are some differences, but most of these won’t
affect the binding sites for inhibitors to the substrate,” he says.

Even though virtual screening makes it possible to discover molecules relatively quickly, these compounds still
need to be experimentally tested — for example, against the virus in cell culture — before any further steps
can be taken. “Unfortunately, with previous outbreaks, there is the experience that not all of these studies are
very sound, they have been done under tight pressure, and so one has to be a little bit cautious about the
results,” says Hilgenfeld.

Targeting viral replication with drugs such as remdesivir should prevent asymptomatic, mild or moderate
COVID-19 cases from progressing to severe disease. Yet current drugs in trials might not be enough for those
who are sickest. “Typically those presenting themselves at hospitals will already have severe disease, which is
associated with pneumonia. Here, targeting viral replication might take away the virus but not the damage
that is very likely due to the immune response of the patient,” says Munster.

The repurposed pipeline is finite, so it’s almost certain that novel as well as repurposed drugs will be needed
in the fight against COVID-19. Those that hold the most promise, Painter notes, are those antivirals with a
breadth of activity against not only coronaviruses, but other RNA viruses, such as highly pathogenic Ebola and
avian flu viruses.. “If there’s any message to be had from the current outbreak, it’s that it’s almost inevitable
it will happen again.”

TABLE 1 | SELECTED REPURPOSED DRUGS IN CLINICAL DEVELOPMENT TO TREAT COVID-19

Originator Clinical trials (trial

Drug or cocktail Status and mechanisms
company posting date)

ASC09 is an experimental
HIV-1 protease inhibitor;
ritonavir and
At least three trials
ASC09/ritonavir, lopinavir/ritonavir are
Ascletis, AbbVie, (e.g.,
lopinavir/ritonavir, with approved protease
Pharmstandard ChiCTR2000029603,
or without umifenovir inhibitors for HIV/AIDS;
2/6/20)
umifenovir is an
approved entry inhibitor
against influenza

ASC09/oseltamivir, See above; oseltamivir is One trial

Ascletis, Gilead,
ritonavir/oseltamivir, a sialidase inhibitor (NCT04261270,
AbbVie
oseltamivir approved for influenza 2/7/20)

Zhengzhou Experimental reverse One trial

Azvudine Granlen transcriptase inhibitor (ChiCTR2000029853,
PharmaTech drug against HIV-1/AIDS 2/15/20)

Baloxavir marboxil is a
Cap-dependent
endonuclease inhibitor Two trials
Various combinations
and favipiravir is a (ChiCTR2000029544,
of baloxavir Shionogi, Toyama
guanine analog RNA- 2/3/20;
marboxil/favipiravir Chemical
dependent RNA ChiCTR2000029548,
and lopinavir/ritonavir
polymerase inhibitor 2/4/20)
approved for influenza A
and B; see above

Darunavir and cobicistat

are, respectively, an HIV-
1 protease inhibitor and
Various combinations Two trials
inhibitor of cytochrome
of darunavir/cobicistat (NCT04252274,
P450 (CYP)3A enzyme,
alone or with Janssen, Gilead 2/5/20;
approved as a
lopinavir/ritonavir and ChiCTR2000029541,
combination against HIV-
thymosin α1 2/3/20)
1/AIDS. Thymosin α1 is
an immune response
boosting agent
 Phosphoramidate
prodrug of an adenine
analog used for Ebola Two trials
and Marburg virus (NCT04252664,
Remdesivir Gilead
outbreaks (similar 2/5/20; NCT04257656,
structure to approved 2/6/20)
HIV reverse transcriptase
inhibitors)

Shanghai Zhongxi
Pharmaceutical, At least ten trials (e.g.,
Chloroquine or Shanghai Ziyuan Endosomal acidification ChiCTR2000029826,
hydroxychloroquine Pharmaceutical, fusion inhibitor 2/2/20; NCT04261517,
Wuhan Wuyao 2/14/20)
Pharmaceutical

Synthetic corticosteroid
that binds to nuclear One trial
Methylprednisolone Generic receptors to dampen (NCT04263402,
proinflammatory 2/10/20)
cytokines

Interferon alfa-2b is a Two trials

Interferon alfa-2b alone
recombinant cytokine (NCT04254874,
or in combination with
Biogen, Merck with antiviral properties; 2/5/20;
lopinavir/ritonavir and
ribavirin is a guanine ChiCTR2000029308,
ribavirin
derivative; as above 1/23/20)

Two trials
Camrelizumab is a
Incyte, Shanghai (ChiCTR2000029806,
Camrelizumab and humanized monoclonal
Hengrui 2/14/20;
thymosin antibody (mAb) targeting
Pharmaceutical NCT04268537,
PD-1
2/14/20)

Chugai
Pharmaceutical,
One trial
Zhejiang Hisun Humanized mAb
Tocilizumab (ChiCTR2000029765,
Pharmaceutical, targeting interleukin-6
2/13/20)
Jiangsu Qyun Bio-
Pharmaceutical

Last search run on 15 February using https://clinicaltrials.gov and http://www.chictr.org.cn.

Excludes traditional Chinese medicines and blood-derived products, such as serum from recovered patients
and stem cells.
All trials are being conducted in China.